Establishment of protoplast preparation protocol and genetic transformation system for Sclerotiophoma versabilis

Going into deep study about the molecular mechanism of pathogenesis in Sclerotiophoma versabilis required high-efficiency transformation system. S. versabilis is a pathogenic fungus that causes severe leaf spot disease in Pseudostellaria heterophylla, a famous traditional Chinese medicinal plant. Here, we report an appropriate system for the production and regeneration of protoplasts with high viability, and then developed a stable and efficient genetic transformation system based on PEG-mediated transformation of protoplasts for S. versabilis . The optimum condition for protoplast preparation is 3 g of the prepared mycelia resuspended in 30 mL 1 M KCl (pH 5.8) containing 25 mg/mL lysing enzyme, incubated at 30 °C and 85 rpm for 3 h. The regeneration rate of the protoplasts was more than 90%. S. versabilis was highly sensitive to hygromycin B and geneticin, but showed high degree of resistance to zeocin. The sGFP gene was successfully transformed into the S. versabilis protoplasts using a modified PEG-mediated transformation method. PCR amplification and southern blot hybridization. GFP signal was detected in both mycelia and conidia of the transformants by confocal microscopy assay. The transformants showed no phenotypic difference from the wild type strain, which furthers the observation of infection process under the fluorescence microscope. The procedures for protoplast isolation, regeneration and transformation provide a foundation for understanding the mechanism of pathogenesis in S. versabilis.